Biology Reference
In-Depth Information
measured, all of which coelute to positively identify the precursor/
peptide of interest [
1
]. A group of transitions per peptide is termed
assay
and is instrument specifi c. Due to the nature of SRM-MS, the
ion chromatogram is distinct from conventional shotgun MS/MS
spectra, which record all possible fragment ions of a particular pre-
cursor ion. Hence, SRM-MS does not depend on a single spec-
trum for positive identifi cation, but on coeluting transitions [
1
]
(
see
Notes 1, 2
and
6
).
Using unscheduled SRM, the number of transitions is defi ned
by the
dwell time
of each transition and the
cycle time
. The cycle
time is defi ned as time it takes to measure all transitions listed in
the method, the dwell time per transition and some instrument
specifi c set up time to switch the
m/z
value of the fi lters. For exam-
ple if each transition is measured for 25 ms dwell time and instru-
ment set up time is 3 ms, in 2,000 ms approximately 71 transitions
can be measured. With 5 transitions per peptide, an unscheduled
SRM-MS experiment would allow to monitor 14 peptides. As this
number is relatively low, scheduled SRM-MS is employed. For
scheduled SRM-MS, prior knowledge of the retention time of a
peptide is required (
see
Note 3
). Effectively, a peptide assay is mea-
sured only during a 2-4 min window during the LC-SRM-MS run
allowing for hundreds of assays to be monitored in a single injec-
tion [
2
] (
see
Note 5
).
Another strength of SRM-MS is the robustness of the method-
ology allowing for consistent measurements of different sample
conditions, but also obtaining reliable results across various labora-
tories with coeffi cient of variation of less than 20 % [
3
] (
see
Note 4
).
The robustness of measurements extends to quantifying analytes
using stable isotope labeled reference standards. Typically, the
endogenous analyte is unlabeled (
see
Note 8
), while the exogenous
reference peptide is marked using stable isotope labeling methods
(
see
Note 7
). As the synthetic reference peptides fragment identi-
cally to the endogenous peptides, the same SRM assay applies,
albeit with the appropriate mass shift. If the relative order of transi-
tions is not conserved between endogenous and exogenous pep-
tides, interference might be the cause (
see
Note 2
).
To guide the reader through the SRM-MS workfl ow, a fl ow-
chart of the SRM-MS method development is shown in Fig.
1
and
explained in detail in each section.
2
Methods
There are multiple software tools utilized in a typical SRM-MS
workfl ow. Table
1
gives an overview of the software tools described
in this chapter with detailed descriptions included in their respec-
tive sections.
2.1
Software Tools
